Protein engineering via directed evolution and gene shuffling has been extensively applied for the systematic improvement of protein properties such as antibody binding affinity, enzyme regulation, and increased or diverse substrate specificity. A similar approach whereby continuously improved mutants are generated along a selection-defined trajectory in the sequence space can also be applied for the systematic improvement or modification of other types of biological sequences, e.g. ribozymes. We show here that promoters can also be engineered via directed evolution to achieve precise strengths and regulation, and, by extension, can constitute libraries exhibiting broad ranges of genetic control.
Typically, the deletion and the strong over-expression of genes have been the principal strategies for elucidation of gene function. These two methods sample the continuum of gene expression at only a few discrete points, determined by experimental feasibility and not necessarily biological significance. Thus, the full dependency of phenotype on gene expression may not be accessible due to the limitations inherent in these methods. Despite prior attempts, no previous work has developed a fully-characterized, homogeneous, broad-range, functional promoter library and demonstrated its applicability to the analysis of such a genetic control.
While inducible promoters allow for a continuous control of expression at the macroscopic level, practical applications of these systems are limited by prohibitive inducer costs, hypersensitivity to inducer concentration, and transcriptional heterogeneity at the single-cell level. The latter factor in particular, can limit the effect of inducers in a culture to a simple increase of the number of cells expressing the gene of interest instead of the overexpression of the gene in all cells. Inducible systems are suitable in certain applications (e.g. recombinant protein overproduction); however, the elucidation of gene function and genetic control on phenotype requires well characterized promoter libraries which are homogeneous at the single cell level.